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Amid et al. Chemistry Central Journal 2012, 6:117
http://journal.chemistrycentral.com/content/6/1/117
RESEARCH ARTICLE
Open Access
Chemical composition and molecular structure of
polysaccharide-protein biopolymer from Durio
zibethinus seed: extraction and purification
process
Bahareh Tabatabaee Amid1, Hamed Mirhosseini1* and Sanja Kostadinović2
Abstract
Background: The biological functions of natural biopolymers from plant sources depend on their chemical
composition and molecular structure. In addition, the extraction and further processing conditions significantly
influence the chemical and molecular structure of the plant biopolymer. The main objective of the present study
was to characterize the chemical and molecular structure of a natural biopolymer from Durio zibethinus seed. A
size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS) was applied to analyze the
molecular weight (Mw), number average molecular weight (Mn), and polydispersity index (Mw/Mn).
Results: The most abundant monosaccharide in the carbohydrate composition of durian seed gum were galactose
(48.6-59.9%), glucose (37.1-45.1%), arabinose (0.58-3.41%), and xylose (0.3-3.21%). The predominant fatty acid of the
lipid fraction from the durian seed gum were palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0),
oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:2). The most abundant amino acids of durian seed
gum were: leucine (30.9-37.3%), lysine (6.04-8.36%), aspartic acid (6.10-7.19%), glycine (6.07-7.42%), alanine
(5.24-6.14%), glutamic acid (5.57-7.09%), valine (4.5-5.50%), proline (3.87-4.81%), serine (4.39-5.18%), threonine
(3.44-6.50%), isoleucine (3.30-4.07%), and phenylalanine (3.11-9.04%).
Conclusion: The presence of essential amino acids in the chemical structure of durian seed gum reinforces its
nutritional value.
Keywords: Biopolymer, Durio zibethinus, Molecular structure, Chemical structure, Carbohydrate, Essential amino acid,
Fatty acid composition
Background
Durian (Durio zibethinus) is the most popular seasonal
fruit in South East Asia countries, particularly Malaysia,
Indonesia, Thailand, and Philippines [1,2]. The botanical
taxonomy of durian brings to light many of the taxonomic problems. Initially, the genus Durio was created
by Rumphius (1741) in his ‘Herbarium Amboinense’.
Later, it was rendered into Linnaean by Adanson [3]. The
type species Durio zibethinus is attributed to ‘Murr’.
However, some researchers attributed the species to
* Correspondence: [email protected]
1
Department of Food Technology, Faculty of Food Science and Technology,
University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Full list of author information is available at the end of the article
Linnaeus (L.) which appeared several times in early taxonomic literature. The earliest valid publication (Murray,
1774) also indicates that the botanical taxonomy of
the species is referred to ‘D. zibethinus Linnaeus (L.).
Willdenow (1800) also listed ‘Durio zibethinus as the species to Linnaeus (L.) [2,3].
There are hundreds of durian cultivars, but there are
only 30 well recognized Durio species. At least nine
species (i.e. D. zibethinus, D. dulcis, D. grandiflorus, D.
graveolens, D. kutejensis, D. lowianus, D. oxleyanus and
D. testudinarum) produce edible durian fruit [2]. Most
cultivars have a common name including a code number
such as Kop (D99), Chanee (D123), Tuan Mek Hijau
(D145), Kan Yao (D158), D24 and D169. However, only
© 2012 Amid et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Amid et al. Chemistry Central Journal 2012, 6:117
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Durio zibethinus is of economic importance and commercially grown cultivar. Only one-third of durian is edible; whereas the seeds (20-25%) are mostly thrown away
after the consumption. Therefore, this crop waste can be
a significant potential source of raw material useful for
the development of value-added products (e.g., seed
gum, flour and etc.).
The term “gum” is used to describe a group of naturally
occurring polysaccharides and/or proteins originated from
different sources (i.e. animal, plant and microbial). Natural
plant gums are usually safe for oral consumption and are
preferred over analogous synthetic gums due to their
safety (non-toxic), low cost and availability [4]. Plant gums
are usually heteropolysaccharide gums composed of simple hexoses and deoxy sugar units such as arabinose, galactose, glucose, mannose, xylose, uronic acids and etc.
The chemical composition and molecular structure of
polysaccharide plant gums play a significant role in their
biological properties. In fact, the functional properties of
polysaccharide plant gums are governed by the chemical
composition, molecular weight, sequence of monosaccharide, configuration of glycoside linkages, and the position
of glycoside linkages in the backbone and side chains [5].
The main goal of the current study was to investigate
the effects of different extraction and purification methods
on the chemical and molecular structure of durian seed
gum. The chemical and molecular structure analysis were
carried out by assessing the sugar composition, moisture,
ash, lipid content, fatty acid composition, molecular
weight (Mw), number average molecular weight (Mn), and
polydispersity index (Mw/Mn ratio). To the best of our
knowledge, there is no similar study reporting the effect of
different extraction and purification processes on the
chemical and molecular structure of durian seed gum.
Results and discussion
Sugar composition of different crude durian seed gums
The sugar analysis revealed that D-galactose (54.4-58.2%)
was the most abundant monosaccharide in the
carbohydrate profile of aqueous- and chemically extracted
gums from durian seed (Table 1, Figure 1). The results
also demonstrated the presence of a high quantity of glucose (40.8-44.6%) in the carbohydrate profile of durian
seed gum. This might be due to the accumulation of
highly water soluble monosaccharide (i.e. glucose) during
the extraction process. The presence of the high glucose
content in the carbohydrate composition of durian seed
gum might be also due to contamination from the seed
coat. Amin and co-workers [6] also demonstrated that
the glucose, galactose, and rhamnose were the main
monosaccharide compositions in the molecular structure
of crude durian seed gum. However, the current study
revealed the presence of a low percentage of rhamnose in
the chemical structure of durian seed gum. This difference might be due to different extraction methods, different experimental conditions of the carbohydrate analysis.
Dawkins and Nnanna [7] also reported a high percentage
of glucose (98.4%) in the gum from oat seed. As reported
by Palanuvej et al. [8], galactose and glucose were the
main monosaccharide in the chemical composition of glucomannan from Litsea glutinosa leaves, Hibiscus esculentus and Scaphium scaphigerum fruits, Ocimum canum,
Plantago ovata and Trigonella foenum-graecum seeds. As
shown in Table 1, the percentage of glucose and galactose
of durian seed gum was higher than that of Yanang
gum [9], locust bean gum [10], malva nut gum [11], and
Prosopis seed gum [12]. Conversely, it showed a lower
percentage of arabinose and xylose than Yanang gum,
locust bean gum, and Malva nut gum (Table 1).
The results also showed the presence of the low
amount of xylose and arabinose and trace amount of
rhamnose in the chemical structure of aqueous- and
chemically extracted gums from durian seed (Table 1,
Figure 1). Palanuvej et al. [8] also reported the presence
of minor content of xylose and arabinose in the chemical
composition of glucomannan from Litsea glutinosa
leaves, Hibiscus esculentus and Scaphium scaphigerum
fruits. As stated by previous researchers [13], arabinose
Table 1 Comparison between sugar composition of durian seed gum and other plant based gums
Plant gum
Monosaccharide composition
Rham
Xyl
Arab
Glu
Gal
Man
Durian seed gum a
trace
0.4 ± 0.1
0.6 ± 0.0
40.8 ± 3.9
58.2 ± 3.2
-
b
Durian seed gum
trace
0.3 ± 0.0
0.8 ± 0.1
44.6 ± 2.8
54.4 ± 3.5
-
Durian seed gum c
21
-
-
70
8
-
Yanang gum d
0.5
72.9
7.7
11.0
8.4
-
0.2 ± 0.1
0.6 ± 0.1
1.9 ± 0.1
4.1 ± 0.1
14.6 ± 0.2
51.9 ± 0.5
29.4 ± 0.1
2.1 ± 0.1
31.9 ± 0.2
2.7 ± 0.2
29.2 ± 0.2
4.8 ± 0.3
2.5 ± 0.2
13.9 ± 1.2
27.3 ± 0.8
56.3 ± 0.8
Locust bean gum
e
Malva nut gum f
Prosopis seed gum g
-
-
Data presented are the mean value ± standard deviation, Rham Rhamnose, Xyl Xylose, Arab Arabinose, Glu Glucose, Gal Galactose, Man Mannose, a Crude
aqueous durian seed gum (current study), b Crude chemically-extracted durian seed gum (current study), c Amin et al. [6], d Singthong et al. [9], e Dakia et al. [10],
f
Somboonpanyakul et al. [11], g Ibaňez and Ferrero [12].
Amid et al. Chemistry Central Journal 2012, 6:117
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Page 3 of 14
of durian seed gum (Table 1). In fact, durian seed
gum does not seem to have mannose; therefore, it is
classified as a glucogalactan, not galactomannan or
arabinogalactan.
Sugar composition of different purified durian seed gums
The results showed that the purification process significantly (p < 0.05) affected the carbohydrate composition of
crude durian seed gum (Figure 2). However, the significant
effect of the purification process on the carbohydrate profile seems to be dependent upon the process condition.
The precipitation using Fehling solution (Method D)
caused the most significant effect of on the percentage of
arabinose; while both purification method A and B had
the least effect on the arabinose content. The results
showed that the purified gum D and B had the highest
and lowest percentage of arabinose among all samples.
The purification using saturated barium hydroxide
(Method C) also induced the significant (p < 0.05) effect
on the percentage of arabinose (Figure 2a).
The current study revealed that the purification
using Fehling solution resulted in the highest significant
Figure 1 HPLC chromatograms showing the sugar profile of
aqueous (a) and chemically (b)-extracted durian seed gums.
is a pentose monosaccharide which contributes to the
molecular structure of various plant gums. Low level of
arabinose is normally occurring as a free sugar in the
side chain of polysaccharide gums; where xylose are
mostly present in the backbone of polysaccharide gum
(arabinoxylan) [13]. According to Da Silva and Gonçalves
[14], the presence of a minor content of arabinose, xylose
and glucose could be attributed to a more complex polysaccharide composition. They also demonstrated that this
may be due to contaminants proceeding from the seed
coat. As stated by León De Pinto and co-researchers [15],
arabinose and xylose are positioned as terminal and terminal residues in the chemical structure of Acacia tortuosa, respectively. In general, the chemically-extracted
durian seed gum had a higher percentage of arabinose
and glucose as well as a lower percentage of xylose
and galactose than the aqueous-extracted durian seed
gum (Table 1). Both durian seed gums had a lower
percentage of xylose than flaxseed gum reported by
previous researchers [16]. Although, the carbohydrate
analysis of durian seed gum confirmed the presence
of high galactose content, but it did not reveal the
presence of mannose in the carbohydrate composition
Figure 2 The percentage of xylose and arabinose (a) as well as
glucose and galactose (b) in crude and different purified durian
seed gums (A: isopropanol and ethanol; B: isopropanol and
acetone; C: saturated barium hydroxide; D: Fehling solution);
Mean ± standard deviation; a-c: Significant (p < 0.05) difference
among samples in terms of xylose and glucose; A-D: Significant
(p < 0.05) difference among samples in terms of arabinose and
galactose.
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This might be explained by the significant (p < 0.05)
effect of the purification process on the carbohydrate
composition of durian seed gum.
(p < 0.05) changes in the monosaccharide composition
(i.e. xylose and arabinose) of durian seed gum (Figure 2).
As stated by previous researchers [17], Fehling solution containing 3M NaOH can promote the chain
degradation of polysaccharide, thereby affecting the
monosaccharide composition of the gum. On the other
hand, the chemical purification using isopropanol and
ethanol (Method A) and isopropanol and acetone
(Method B) did not significantly change the percentage of
xylose present in the carbohydrate composition of durian
seed gum (Figure 2a). However, both purified gums A and
B had the lower xylose content than the crude gum
(Figure 2a). Da Silva and Gonçalves [14] also reported that
the purified locust bean gum showed a lower content of
xylose than its crude gum. The significant changes in
xylose content may significantly influence the rheological
properties of the gum. As stated by previous researches
[18], xylose content reflects the relative amount of neutral polysaccharides, which enhances rheological properties of the gum by increasing the shear thinning behavior
and weak-gel properties.
As shown in Figure 2b, the purified gum D and A
showed the highest and lowest percentage of glucose
among all purified gums. In general, all purification processes (except for Method D) significantly (p < 0.05)
reduced the percentage of glucose in durian seed gum. The
level of glucose reduction was dependent upon the purification technique as follows: purified gum A > purified
gum B > purified gum C > crude gum > purified gum D
(Figure 2b). Previous researchers [14,19,20] also reported
the glucose reduction after purifying the crude locust bean
gum, fenugreek gum and guar gum, respectively.
As shown in Figure 2b, the purified gum A (isopropanol
and ethanol) and B (isopropanol and acetone) showed a
higher percentage of galactose than the crude gum.
Previous researchers [19] also reported the similar observation for the crude and purified fenugreek gum. They
found that the purified fenugreek gum had a higher percentage of galactose than the crude fenugreek gum. The
results indicated that the purified gum A and D showed
the highest and lowest percentage of galactose among all
samples (Figure 2b). Our previous study [21] reported
the significant (p < 0.05) different rheological properties
and viscoelastic behavior of the crude and purified gums.
Moisture and ash content
The chemical extraction method resulted in lower moisture content than the aqueous extraction method. On
the other hand, there was a significant (p < 0.05) different
between the moisture content of the crude and purified
durian seed gums. In general, all purification methods
led to reduce the moisture content as compared to both
crude gums (Table 2). The purified gum A and C had the
highest and lowest significant moisture content among
all purified gum. However, the purified gum A had higher
moisture content than the crude gum (Table 2). Durian
seed gum had a relatively high content of moisture (20.526.8%) in both crude and purified forms (Table 2). This
value was greater than the moisture content reported for
corn fiber gum (4.0-5.9%) [22], guar gum (7.36%), locust
bean gum (7.92%), and gum karaya (9.43%) [23], acacia
glomerosa gum (11.29) [24], grewia gum (10.6- 18.8) [25],
and Baobab leaves gum (7.8-8.0) [26] (Table 3).
The total ash is a useful figure for determining the
characterization and purity of the gum [27]. This parameter gives an indication of the degree of mineral interaction in the structure which contributes to the functional
properties of the polysaccharide gum. The lower ash content is associated with higher purity degree [27]. The
results indicated that the purification process significantly
(p < 0.05) decreased the ash content of durian seed gum.
On the other hand, the purification significantly (p < 0.05)
affected the soluble ash content as compared to the crude
gum (Table 2). The degree of changes depended on the
purification condition (Table 2). The current study
revealed that durian seed gum had a relatively high content of ash (12.1 ± 34.3%). This value was higher than the
ash content reported for Africana seed gum (3.63-3.65%)
[24], corn fiber gum (4.0-5.9) [28], gum Arabic (1.2%),
guar (11.9%) [16], grewia gum (6.1 -6.3%) [25], okra fruit
gum (4.81-5.95%), and baobab leaves gum (8.8-9.88%) [26]
(Table 3). Amin and co-researchers [6] also reported a
relatively high total ash (29.8%) in durian seed gum. The
results also indicated that both crude and purified durian
seed gums had relatively low content of soluble ash
Table 2 Moisture, ash and lipid content of durian seed gum
Test
Crude gum (Aqueous)
Crude gum (Chemical)
Purified gum A
Purified gum B
Purified gum C
Purified gum D
Moisture %
26.8 ± 1.09a
24.6 ±1.22ab
23.2 ± 1.03b
22.8 ± 0.79bc
20.5 ± 0.35c
21.7 ± 1.13bc
Total ash %
a
a
b
Soluble ash %
Lipid %
32.8 ± 1.57
a
1.7 ± 0.03
a
1.92 ± 0.07
34.3 ± 1.78
a
1.5 ± 0.01
b
0.78 ± 0.11
20.6 ± 0.89
b
0.9 ± 0.00
c
0.14 ± 0.04
b
23.4 ± 1.34
b
1.0 ± 0.14
c
0.16 ± 0.02
c
12.1 ± 0.94d
c
0.5 ± 0.06d
d
0.19 ± 0.03d
15.8 ± 1.13
0.7 ± 0.03
0.21 ± 0.06
Data presented are the mean value ± standard deviation, A isopropanol and ethanol, B isopropanol and acetone, C saturated barium hydroxide, D Fehling
solution, a-d significant differences at 95% confident level.
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Table 3 Moisture, ash, and molecular weight (Mw) of
various plant gums
Sample
Moisture %
Ash
%
Soluble
ash%
Mw
Acacia glomerosa gum 1
11.29
7.98
-
0.9 × 105
Corn fiber gum 2
4.0-6.6
4.0-5.9
-
-
10.6- 18.8
6.1 -6.3
3.4 -3.8
-
9.35-9.37
4.81-5.95
-
-
7.8-8.0
8.8-9.88
-
> 1.0 ×105
Grewia gum
3
Okra fruit gum 4
Baobab leaves gum 4
1
4
Gundidza et al. [24]; 2 Yadav et al. [22]; 3 Nep and Conway [25];
Woolfe et al. [26].
content, ranging from 0.5 to 1.7% (Table 2). The purified
gum D followed by purified gum C had the lowest
content of soluble ash; while the aqueous and chemically
extracted crude gums provided the highest soluble ash
among all samples (Table 2). This might indicate the efficiency of all purification processes to reduce the gum
impurities.
Lipid content and fatty acid composition
The results indicated that different crude and purified
durian seed gums had significant (p < 0.05) different
lipid content, ranging from 0.19 to 1.92% (Table 2). A
comparative lipid analysis of different crude and purified
durian seed gums showed that the purification process
significantly (p < 0.05) reduced the lipid content, thus
enhancing the gum purity (Table 2). However, none of
purification techniques enabled to completely remove
the lipid fraction present in the chemical structure of the
crude durian seed gum. This could be explained by the
fact that the lipid fraction might be a part of chemical
structure of durian seed gum. Yadav and co-researchers
[22] reported the presence of the lipid fraction as a part
of the molecular structure of gum Arabic, inducing the
emulsifying activity. In addition, the presence of the
lipid fraction in the chemical structure of many polysaccharide gums have been reported by previous researchers
[22,28,29].
As mentioned earlier, both aqueous and chemically
extracted durian seed gum contained a relatively higher
content of lipid fraction than the purified gums (A-D).
Our previous study [30] revealed that the crude gum
from durian seed showed the interfacial activity in oil/
water (o/w) emulsion system. This might be due to the
presence of trace amount of the hydrophobic lipid fraction along with the hydrophilic polysaccharide fraction
present in the chemical structure of durian seed gum.
However, the emulsifying activity of durian seed gum
might be also due to the presence of the protein fraction
present in the chemical structure of durian seed gum
may also contribute to its emulsifying activity [30].
The chemically-extracted crude gum showed a significant
(p < 0.05) lower fat content (0.78 ± 0.11) than the
aqueous crude gum (1.92 ± 0.07). This might be due to
the defatting process occurred during the chemical extraction process. In fact, durian seed was defatted and
discolored by using organic solvent during the chemical
gum extraction, thus more efficiently reducing the lipid
fraction impurity than the aqueous extraction process.
This indicates the less efficiency of aqueous extraction
technique than the chemical extraction method to reduce
the hydrophobic impurities (i.e. lipid fraction).
On the other hand, the significant different between
the lipid content of aqueous- and chemically-extracted
durian seed gums might be responsible for their significant different levels of solubility. The results indicated
that the precipitation using isopropanol and ethanol
(Method A) looks to be the most efficient purification
technique for the removal of the hydrophobic lipid
fraction from the crude durian seed gum (Table 2).
Conversely, the precipitation using saturated barium
hydroxide seems (Method C) seems to be the least efficient purification technique for reducing the hydrophobic impurity (i.e. lipid fraction) of the crude durian seed
gum (Table 2). As also shown in Table 2, the purified
gum A and C had the lowest and highest content of the
lipid fraction among all purified gums.
Figure 3 shows the fatty acid composition of the lipid
fraction from (a) aqueous extracted crude gum, (b)
chemically-extracted crude gum, (c) purified seed gum A,
(d) purified seed gum B, (e) purified seed gum C, and
(f ) purified seed gum D. The predominant fatty acid of
the lipid fraction obtained from the crude and durian
seed gums were palmitic acid (C16:0), palmitoleic acid
(C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic
acid (C18:2), linolenic acid (C18:2), and arachidic acid
(C20:0) (Table 4). Previous researchers [28] also reported
the presence of palmitic acid (C16:0) and oleic acid
(C18:1) in the lipid fraction extracted from corn fibre
gum. The current study revealed that the lipid fraction
from durian seed had a higher amount of saturated fatty
acid (SFA) (C18:0, C18:0 and C20:0) rather than unsaturated fatty acid (C16:1, C18:1, C18:2 and C18:3).
As shown in Table 4, the aqueous-extracted gum
seems to have the highest content of saturated fatty acid
(SFA) (C18:0, C18:0 and C20:0), monounsaturated fatty
acid (MUFA) (C16:1 and C18:1), and polyunsaturated
fatty acid (PUFA) (C18:2 and C18:3) among all crude
and purified gums (A-D). In most cases, the purified
gum D had the highest percentage of SFA, MUFA and
PUFA among all purified sample; while the purified gum
A showed the lowest significant (p < 0.05) content of
C18:0, C18:1, C18:2, C18:3 and C20:0 among all samples
(Table 4). The purification using isopropanol and ethanol
(Method A) and isopropanol and acetone (Method B)
seems to have more efficiency than the precipitation using
saturated barium hydroxide (Method C) and Fehling
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Figure 3 Fatty acid profile of the lipid fraction from (a) aqueous crude gum, (b) chemical crude gum, and (c-f) purified seed gums
(a-d); C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:2 (linolenic acid),
and C20:0 (arachidic acid).
solution (Method D) for reducing SFA, MUFA and PUFA
present in the chemical structure of durian seed gum.
Vinod and co-researchers [31] reported the presence
SFA such as capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid
(C18:0), arachidic acid (C20:0), behenic acid (C22:0), and
lignoceric acid (C24:0) in the fatty acids composition of
gum kondagogu. They found that stearic acid (25.4 ±
1.54 lg/g) was the main saturated fatty acid present in
gum kondagogu. In addition, the researchers reported
that the presence of mono- and polyunsaturated fatty
acids such as palmitoleic acid (C16:1, 7.9 ± 0.54 lg/g),
oleic acid (C18:1, 5.1 ± 0.18 lg/g), erucic acid (C22:1,
9.9 ± 0.54 lg/g), linoleic acid (C18:2, 1.8 ± 0.12 lg/g),
Table 4 Fatty acid composition present in the chemical structure of durian seed gum
Fatty acid a
Gum
C16:0
Crude gum
1
Crude gum
2
C16:1
a
642 ± 57
b
337 ± 42
cd
C18:0
52 ± 9
33 ± 2
b
138 ± 12
c
c
Purified gum A
141 ± 36
15 ± 3
Purified gum B
116 ± 19c
21 ± 5c
Purified gum C
Purified gum D
cd
158 ± 31
d
203 ± 46
C18:1
a
11 ± 3
c
24 ± 4
c
a
287 ± 17
b
76 ± 9
C18:2
a
374 ± 28
d
108 ± 16
cd
101 ± 15
75 ± 11
423 ± 39a
36 ± 4
b
151 ± 18b
14 ± 3
c
62 ± 5c
158 ± 26
c
c
169 ± 23
107 ± 10d
cd
99 ± 14
bd
141 ± 17
76 ± 8
C20:0
b
346 ± 48
b
63 ± 11
93 ± 13cd
C18:3
a
103 ± 16bc
c
95 ± 13
b
146 ± 21
a
17 ± 3c
76 ± 12c
25 ± 6
bc
69 ± 7c
31 ± 5
b
137 ± 23b
a
In μg/g of the dry weight of samples; Data presented are the mean value ± standard deviation; 1: Aqueous crude gum; 2: Chemically extracted crude gum; C16:0,
palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; A: isopropanol and ethanol; B: isopropanol and acetone; C: saturated
barium hydroxide; D: Fehling solution.
Amid et al. Chemistry Central Journal 2012, 6:117
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linolenic acid (C18:3, 0.8 ± 0.05 lg/g) in gum kondagogu.
The same researchers [31] investigated the fatty acid
composition of gum karaya. They reported that the major
fatty acids of gum karaya were capric acid (C10:0), lauric
acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0),
palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid
(C18:1), and behenic acid (C22:0). The most abundant
unsaturated fatty acids present in the chemical structure
of gum karaya were palmitoleic acid (13.2 ± 0.95 lg/g),
oleic acid (4.2 ± 0.21 lg/g), and erucic acid (2.8 ± 0.12 lg/g)
[31]. The previous study [31] showed that gum karaya
had higher content of SFA and lower content of PUFA
than gum kondagogu. Initially, it was hypothesized that
the fatty acid profile might be a reliable indicator to discriminate various plant gums from each other. However,
Page 7 of 14
the fatty acid composition of the gum is significantly
affected by the extraction, purification and further processing condition; therefore this parameter cannot be an accurate tool to differentiate various plant gums from each
other.
Amino acid composition
The amino acid composition can be a useful feature for
the chemical characterization of the heteropolysaccharide
gum. This parameter directly contributes to the solubility
and functional properties of the heteropolysaccharideprotein gum. Our previous study [30] revealed the presence of the protein fraction in the chemical structure of
durian seed gum. Figure 4 (a-e) demonstrates HPLC
Figure 4 Amino acid profile of (a) crude gum, (b) purified gum a, (c) purified gum b, (d) purified gum c, (e) purified gum d; aspartic
acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro),
internal standard (IS, alpha amino butyric acid), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu),
phenylalanine (Phe), and lysine (Lys).
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Page 8 of 14
chromatograms which show the amino acid composition
of the protein fraction present in the chemical structure of
durian seed gum. The current study revealed that the
most abundant amino acid present in the chemical structure of durian seed gum were leucine (Leu) (30.9-37.3%),
phenylalanine (Phe) (3.11-9.04%), lysine (Lys) (6.048.36%), glycine (Gly) (6.07-7.42%), aspartic acid (Asp)
(6.10-7.19%), glutamic acid (Glu) (5.57-7.09%), alanine
(Ala) (5.24-6.14%), threonine (Thr) (3.44-6.50%), valine
(Val) (4.5-5.50%), serine (Ser) (4.39-5.18%), proline (Pro)
(3.87-4.81%), and isoleucine (Ile) (3.30-4.07%) (Table 5).
In addition, the amino acid analysis also revealed the
presence of a minor quantity of methionine (Met) (0.500.98%), histidine (His) (0.81-1.14%), arginine (Arg) (2.383.51%), and tyrosine (Tyr) (1.63-2.19%) in the amino
acid composition of durian seed gum (Table 5). The
presence of a negligible content of histidine, methionine
and tyrosine was also reported by previous researchers in
flaxseed gum [16], Spondias gums [32], and Acacia glomerosa gum [33]. They also reported different contents
of leucine, phenylalanine, lysine, aspartic acid, glycine, alanine, glutamic acid, valine, proline, and serine in various
plant gum such as flaxseed gum [16], Spondias gums
[32], Acacia glomerosa gum [33], and Prosopis gum [34].
Vinod and co-researchers [31] reported that the most
abundant amino acids in gum kondagogu were Ala (32.2
± 1.44 lg/g), Gly (5.05 ± 0.55 lg/g), Val (7.2 ± 0.60 lg/g),
Leu (3.8 ± 0.22 lg/g), Pro (42.4 ± 2.56 lg/g), Met (44.2 ±
2.25 lg/g), Asp (72.8 ± 3.45 lg/g), Thr (30.4 ± 1.54 lg/g),
Tyr (32.8 ± 1.85 lg/g), and Try (10.8 ± 0.84 lg/g).
They also reported that the main amino acids in the
chemical structure of gum karaya (lg/g) included Gly
(4.8 ± 0.45 lg/g), Leu (3.9 ± 0.28 lg/g), Pro (30.5 ±
1.86 lg/g), Asp (64.2 ± 2.44 lg/g), Thr (25.2 ± 1.06 lg/g),
and Glu (34.2 ± 1.44 lg/g). The most abundant amino
acids were Asp (64.2 ± 2.44 lg/g) and Glu (34.2 ±
1.44 lg/g) [31]. The current study revealed that the amino
acid composition of durian seed gum was different from
that of reported for Spondias gum, Prosopis gum, gum
kondagogu, flaxseed gum, gum karaya, gum Arabic,
mesquite gum and guar gum [16,32-34]. For instance,
previous researchers reported the presence of hydroxyproline (Hpr) in the chemical structure of Spondias gum,
Acacia gum, Mesquite gum and Prosopis gum; while
there was no evidence showing the presence of Hpr in
the chemical structure of durian seed gum (Table 5).
Molecular characteristics
The results showed that both extraction and purification
processes significantly (p < 0.05) influenced the molecular
structure of durian seed gum (Table 6). The results indicated that the molecular weight of crude and purified gum
ranged from 1.08 × 105 to 1.44 × 105 (g/mol) (Table 6).
As reported in our previous study [21], different purified
durian seed gums showed significant (p < 0.05) different
rheological behavior and viscoelastic properties. This
might be due to the difference between the molecular
weight of different crude and purified durian seed gums
Table 5 Amino acid composition of the protein fraction from durian seed gum
Amino acid
Asp
Glu
Control sample
a
7.19 ± 0.13
a
7.09 ± 0.22
b
6.19 ± 0.44
b
5.57 ± 0.25
ab
6.95 ± 0.26
a
6.54 ± 0.34
Purified gum D
b
6.50 ± 0.75ab
ab
6.42 ± 0.34a
a
6.10 ± 0.58
6.18 ± 0.35
5.06 ± 0.32
4.53 ± 0.57
5.18 ± 0.12
4.39 ± 0.29a
Gly
7.42 ± 0.29a
6.22 ± 0.58b
6.43 ± 0.20b
7.36 ± 0.20a
6.07 ± 0.15b
His
a
a
a
a
0.81 ± 0.10a
a
2.38 ± 0.22b
bc
a
3.51 ± 0.25
a
1.14 ± 0.19
a
3.40 ± 0.21
0.88 ± 0.04
ab
2.87 ± 0.12
3.16 ± 0.16
6.50 ± 0.19
5.41 ± 0.42
6.47 ± 0.45
4.99 ± 0.43
3.44 ± 0.34c
Ala
6.14 ± 0.36a
5.68 ± 0.24a
5.97 ± 0.23a
6.08 ± 0.25a
5.24 ± 0.43a
Pro
a
a
a
a
4.06 ± 0.58a
a
4.17 ± 0.75
1.91 ± 0.09
1.63 ± 0.22
1.95 ± 0.19
1.84 ± 0.29a
Val
5.15 ± 0.43a
4.59 ± 0.43a
4.50 ± 0.24a
5.50 ± 0.46a
4.90 ± 0.16a
a
a
a
b
0.69 ± 0.09a
a
3.73 ± 0.22a
ab
Ile
a
3.89 ± 0.18
a
3.63 ± 0.22
a
3.30 ± 0.29
4.07 ± 0.23
37.3 ± 1.67
30.9 ± 1.87
34.6 ± 1.87
35.6 ± 1.15a
Phe
3.11 ± 0.12a
9.04 ± 0.87b
6.93 ± 0.22c
8.13 ± 0.38b
7.62 ± 0.26bc
a
a
Hpr
-
7.88 ± 0.46
-
b
0.98 ± 0.10
37.1 ± 1.87
8.36 ± 0.23
a
0.52 ± 0.06
Leu
Lys
a
0.50 ± 0.03
a
4.81 ± 0.42
2.19 ± 0.21
0.59 ± 0.07
a
3.87 ± 0.41
Tyr
Met
a
a
1.00 ± 0.09
Thr
4.35 ± 0.16
b
a
Purified gum C
4.67 ± 0.50
1.13 ± 0.15
a
Purified gum B
Ser
Arg
a
Purified gum A
ab
7.65 ± 0.65
-
a
7.32 ± 0.28
-
6.04 ± 0.25b
-
Aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), tyrosine (Tyr), valine
(Val), methionine (Met), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), and lysine (Lys); A: isopropanol and ethanol; B: isopropanol and acetone; C: saturated
barium hydroxide; D: Fehling solution; a-d significant at p < 0.05: Mean ± standard deviation.
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Page 9 of 14
Table 6 Molecular weight, and polydispersity index (Mw/Mn) of durian seed gum
Test
Crude gum
(Aqueous)
Crude gum
(Chemical)
Purified gum A
Purified gum B
Purified gum C
Purified gum D
Mwt (g/mol)
1.37 × 105 ± 0.024a
1.44 × 105 ± 0.024b
1.26 × 105 ± 0.015c
1.23 × 105 ± 0.019c
1.14 × 105 ± 0.026d
1.08 × 105 ± 0.009e
Mw/Mn
1.34
1.39
1.29
1.26
1.21
1.17
Rg (nm)
28
31
25
23
18
15
Mwt: Molecular weight; Mw/Mn: Molecular weight/number average molecular weight (or polydispersity index); A: isopropanol and ethanol; B: isopropanol and
acetone; C: saturated barium hydroxide; D: Fehling solution.
(A-D). As shown in Table 6, all purification processes led
to reduce the molecular weight. Da Silva and Gonćalves
[14] also showed that the purified locust bean gum (LBG)
had lower molecular weight than the crude LBG. They
reported that the purification of locust bean gum (LBG)
using isopropanol led to reduce the molecular weight as
compared to the crude LBG.
The results indicated that the purification using isopropanol with ethanol (Method A) resulted in the least significant reduction in the molecular weight of durian seed
gum (Table 6). On the other hand, the precipitation using
Fehling solution (Method D) resulted in the highest significant (p < 0.05) reduction in the molecular weight of
durian seed gum. In fact, the purified gum A and D had
the highest and lowest molecular weight among purified
samples (Table 6). This might be explained by the presence of NaOH in Fehling solution which probably
enhanced the degradation of the polysaccharide structure. In addition, the copper complex present in Fehling
solution may cause the cleavage in polysaccharide chains,
thereby reducing the molecular weight of the purified
gum D as compared to the crude gum. In addition, the
removal of the lipid and/or protein fraction from the
gum structure might be also responsible for the further
reduction of the molecular weight during the purification
process.
Different crude and purified durian seed gum had significant (p < 0.05) different Rg values ranging from 15 to
31 nm (chemically-extracted durian seed gum) (Table 6).
The chemically-extracted durian seed gum and purified
durian seed gum D showed the highest and lowest Rg
values (31 nm and 15 nm), respectively (Table 6). The
results indicated that the polydispersity index (Mw/Mn) of
durian seed gum ranged between 1.17 and 1.39 (Table 6).
The polydispersity index (PDI, Mw/Mn) of durian seed
gum significantly decreased after the purification process.
This might be interpreted by the significant reduction
of molecular weight after purifying the crude gum. The
purified gum with lower PDI has more homogeneous
structure than the crude gum with higher PDI. The
high polydispersity value for the crude durian seed gum
might be due to the presence of molecules with a broad
spectrum of molecular weights. The heterogeneous structure with a wide spectrum of molecular weights resulted
in some difficulties in the precipitation process.
Conclusions
The present study revealed that all purification processes
led to reduce the lipid fraction. However, they did not
completely eliminate the lipid fraction from the chemical
structure of the gum. Among all purification techniques,
the precipitation using Fehling solution induced the most
significant (p < 0.05) changes in the chemical composition and molecular structure of the heteropolysaccharide
gum from Durio zibethinus Murray seed. The purified
gum D (using Fehling solution) had the least contents of
total ash, soluble ash, and lipid fraction among all crude
and purified gums. It seems that the purification using
Fehling solution can provide the purified durian seed
gum with the highest purity degree (or least impurity
content) among all studied purification techniques. The
present study revealed the presence of some essential
mono- and polyunsaturated fatty acid (e.g. oleic acid,
linoleic acid, and linolenic acid) in the chemical structure
of durian seed gum. The presence of essential amino acids
(i.e. phenylalanine, valine, tryptophan and isoleucine) in
the chemical structure of durian seed gum reinforces its
nutritional value as compared to amino acid free gums.
The present study recommends testing the biological and
nutritional aspects of the natural biodegradable biopolymer from durian seed.
Experimental
Chemicals and materials
The sugar standards (i.e. L-arabinose, D-(+)-galactose,
D-(+)-mannose, L-rhamnose monohydrate, D-(+) glucose,
D-(−)-fructose and sucrose) were purchased from SigmaAldrich (St. Louis, MO, USA). Acetonitrile (HPLC grade),
sulfuric acid (nitrogen free), ammonium acetate, sodium
hydroxide (NaOH) and hydrochloric acid (HCl) (37%)
were supplied by Merck (Darmstadt, Germany). In this
study, amino acid standards AAS18 including L-alanine
(Ala), L-glycine (Gly), L-valine (Val), L-leucine (Leu),
L-isoleucine (Ile), L-threonine (Thr), L-methionine, (Met),
L-aspartic acid (Asp), L-glutamine (Gln), L-phenylalanine
(Phe), L-tyrosine (Tyr), L-tryptophan (Trp) and hydroxyproline (Hpr) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Ethanol 95%, absolute ethanol
(99.9%), saturated barium hydroxide, acetic acid, hexane,
petroleum ether (40–60°C) and methanol (HPLC grade)
were purchased from Fisher Scientific (Pittsburgh, PA,
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USA). Durian (D. zibethinus) fruit was purchased from
the local market (Selongor, Malaysia). Ripened durian
fruits were selected based on the size uniformity and free
of visual defects. The fruits were then de-husked (cut
open the rind), by cutting along the suture on the back
of the lobules. Durian seeds were removed, cleaned and
rinsed thoroughly with sterile distilled water. The seed
was partially dried by the air circulation. The dried
seeds were then packed in plastic bags and stored in
a dry and cool place (10 ± 2°C) until the extraction
process. All the experiments were performed with
deionized water [35].
Extraction process
Aqueous extraction
The aqueous extraction of the crude gum from durian
seed was carried out in triplicate according to the
method described earlier [35]. In this method, durian
seeds were washed, chopped and ground into the powder
form. Then, the aqueous extraction process was performed by using deionized water from durian seed powder. The aqueous extraction was carried out by using
water: seed (W/S) ratio of 60:1, extraction temperature
40°C under pH 7. In this experiment, pH was continuously adjusted by 0.1 (mol/L) NaOH and HCl. The heat
treatment was performed indirectly by using an adjustable water bath. It avoids the thermal degradation of the
sample which may be occurred by the direct heat treatment. In fact, the water temperature of the adjustable
water bath was controlled before the seed powder was
added to the beaker. The seed-water slurry was stirred
throughout the entire extraction period (1 h, based on
our preliminary tests). The slurry was centrifuged at
1200 rpm for 10 min by using a Beckman Coulter Centrifuge (Avanti J-25, Beckman Coulter GmbH, Krefeld,
Germany). Subsequently, the supernatant was collected.
The supernatant was mixed with three volumes of 95%
ethanol and the percipient was dispersed in deionized
water and oven dried at 40°C [35].
Chemical extraction
The chemical extraction was performed according to the
method described in the previous study [36]. Durian
seed were washed and chopped into small pieces. Then,
it was air dried by using the air circulation before milling
into flour. The cold extraction was used to extract the
oil from durian seed flour in order to avoid the thermal
degradation. The defatting process was carried out successively using hexane and isopropanol (60:40) at the
room temperature (25 ± 1°C). Our preliminary study
showed that the solvent mixture containing hexane and
isopropanol (60:40) was the most efficient solvent for
defatting process among all studied solvents (i.e. petroleum ether, hexane, isopropanol and ethanol). The solvent
Page 10 of 14
residue was removed by centrifugation at ~3000 rpm for
15 min using the Beckman Coulter Centrifuge (JA-14,
Beckman Coulter GmbH, Krefeld, Germany). Then,
defatted-durian seed flour (1 kg) was exhaustively decolored using ethanol at the decoloring time 120 min. The
decolorized seed flour was vacuum filtered and then
soaked in 1% aqueous acetic acid for 1.5 h at the room
temperature (25 ± 1°C). Then, the slurry was filtered with
Nylon cloth filter and the filtrate was precipitated with
95% ethanol. The precipitated slurry was washed three
times using absolute ethanol (99.9%) to achieve very light
brown amorphous crude gum. The crude gum was collected and oven dried at 40°C [36]. The effectiveness of
four different purification techniques was determined by
considering the crude seed gum as a control sample.
Purification process
In the purification Method A, the crude seed gum was
purified by using hot water, ethanol and isopropanol as
described by previous researchers [19,37]. Initially, the
gum solution (2.5% w/v) was prepared by dissolving 25 g
of the crude durian seed gum in 1 l of deionized water
at 80°C water bath for 6 h, followed by stirring at room
temperature overnight. The gum solution (2.5% w/v) was
subjected to the centrifugation for 15 min at ~10000 rpm
using the Beckman Coulter Centrifuge (JA-14, Beckman
Coulter, Krefeld, Germany). The supernatant was precipitated by the addition of absolute ethanol (1.2 l). Then,
the supernatant was decanted, and the residue was recovered and kept overnight in 100% isopropanol. Finally,
the residue was dried in the oven 40°C for overnight to
prepare the purified durian seed gum.
In the purification Method B, the purification process
was carried out by using isopropanol and acetone as
reported by previous researchers [38] with the minor
modification. One g of the crude seed gum was precipitated by soaking into 200 ml isopropanol, and
allowing the gum-solvent slurry to stand for 30 min.
The fibrous precipitate was collected by the filtration
using screen with mesh 53 μm. Then, the collected
precipitate was washed twice with isopropanol and
acetone [21]. Finally, it was dried in the oven
overnight at 40°C.
In the purification Method C, the crude seed gum was
purified through barium complexing according to the
method described by previous researchers [39]. In this
method, the gum solution (2.5% w/v) was prepared by
dissolving 2.5 g of the crude durian seed gum in 100 ml
of water and continuous stirring for 12 h at 60°C. Then,
the gum solution was precipitated with saturated barium
hydroxide solution. The precipitate was separated by the
Beckman centrifuge at 3500 rpm for 15 min. Then, the
precipitate was stirred with 1 M acetic acid for 8 h and
again centrifuged. The supernatant was precipitated with
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90% ethanol. Finally, the precipitate was washed with
95% ethanol and oven dried at 40°C [37].
In the purification Method D, the purification was performed by using Fehling solution as reported by previous
researchers [20] with some modification. Initially, 1 g of
the crude durian seed gum was dissolved in approximately 100 ml of water and stirred for 24 h with magnetic stirring. The prepared gum solution (1% w/v) was
precipitated by adding 5 ml of freshly prepared Fehling
solution, and the precipitate was collected on a glass filter (No. 3). Then, the precipitate was dissolved in 0.1 M
hydrochloric acid while under magnetic stirring for 1 h
until the full solubilisation. The solution was precipitated
with 95% ethanol (120 ml). The precipitate was separated
by the glass filter (No. 3) and washed with 95% ethanol
until pH 6 [37]. Finally, the filtrate was washed with acetone and oven dried at 40°C overnight.
Analytical test
Determination of sugar composition
The sugar composition was determined according to the
method reported by Amin and co-workers [6] with minor
modifications. D-fructose was considered as an internal
standard in the present study. The carbohydrate profile
was determined by using a high performance liquid chromatography (HPLC) (Waters 486, CA, USA) equipped
with a refractive index (RI) detector and a pump Waters
600 controller. The analytical column was Lichrocart
250–4,6 purospher star NH2 column (5 MYM). The
mobile phase composed of acetonitrile–water (75:25).
The flow rate was ranged between 0.4–1.5 ml/min. A
10 mg of crude durian seed gum was degraded by heating
at 80°C for 24 h along with 2 ml of 1 M sulfuric acid.
Next, the hydrolyzed gum was placed under the rotary
evaporator for 4 h at 40°C under the vacuum condition.
Then, 1 ml of deionized water was added to the remaining
gum solution. Finally, the slurry was filtered by using
Sep-Pak cartridges (Waters Associates, Milford, MA,
USA) to remove the phenolic compounds. The filtrated
slurry was passed through a membrane filter of 0.45 μm
(Whatman) before injecting to HPLC system. The sugar
analysis was carried out in triplicate for each sample. For
quantitative analysis of the sugar composition, the following standards were considered: L-arabinose, D-galactose,
D-mannose, D-xylose, L-rhamnose, glucose, fructose (IS)
and sucrose.
Moisture content
The moisture content was determined by using the
method described by previous researchers [6]. Approximately, 2 g of durian seed gum was weighed in the crucible and then placed in the oven at 100°C for 6 h. After
drying at 100°C, the dried sample was covered, cooled
in the desiccator and weighted, accordingly. The gum
Page 11 of 14
samples were taken out and weighed after every 10 min
interval time. This was repeatedly done until the weight
of the samples remained constant. The moisture content was measured in triplicate and estimated based on
the following equation:
% Dry Content ¼
Weight of dry sample
100
Original weight of sample
% Moisture content ¼ 100 % dry content
ð1Þ
Ash content
The total ash was determined according to AOAC Official Method 923.03. [40]. Approximately 5 g of durian
seed gum was weighed into a shallow ash dish. The dish
containing the gum was ignited, cooled in desiccator and
weighed after cooling to the room temperature. Then,
the sample was ignited in a furnace at 550°C (dull red)
until the weight become constant. In this condition, light
gray ash was observed in the dish. Then the gray ash
was weighed after the sample was cooled to the room
temperature in the desiccator. The total ash content was
calculated according to the following equation:
% total ash ¼
ash weight
100
original sample weight
ð2Þ
Soluble ash content was determined by mixing the
total ash with 25 ml distilled water, and the solution was
heated to boil. Then the solution was filtered and soluble
ash was rinsed using the distilled water until the volume
was about 60 ml. The filter paper and its residue were
placed back into the original crucible, where the dish
containing the gum was ignited in this step. The crucible
was placed in the oven again at 550°C until the constant
weight, then cooled and weighed. The ash content was
measured in triplicate for each sample. The soluble ash
was calculated according to the following equation:
% soluble ash ¼ % total ash % insoluble ash
ð3Þ
Lipid content
The lipid extraction was carried out by hydrolyzing durian seed gum according to the method reported by previous researchers [41,42] with minor modification. For
hydrolysis purpose, 1 g durian seed gum was placed in a
screw cap glass tube (55mL, 25mm × 150mm), and then
10 ml of 1.5 M methanolic KOH and 500 μl water were
added to dissolve the sample. The tubes were sealed with
Teflon lined screw caps. Subsequently, the tubes were
immersed in a water bath at 70°C for 1 h under stirring
mode. The tubes were gradually cooled to the room
temperature. Then, 6 ml methanol and 8 ml chloroform
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were added to the tubes and mixed well. The tubes were
subjected to the centrifugation at 70 × g for 15 min and
filtered through a Whatman GF/A glass filter paper
(Whatman Laboratory Products, Clifton, NJ, USA) fitted
in a Buchner funnel under the vacuum condition. The
pellet in the glass tube was suspended in 2 ml of methanol
and chloroform (2:1). The tube was mixed well and filtered through the same filtration set up to collect the
filtrate in the same tube. The filter paper was rinsed with
1 ml of 2:1 methanol and chloroform (2:1) and the filtrate
was collected in the same tube. Subsequently, 8.5 ml of
water was added to the filtrate containing 18 ml methanol and 9 ml chloroform. Then, 6 M HCl was used to
acidify the solution (pH 2–3) and 9 ml chloroform was
added to the solution to maintain the ratio of methanol,
chloroform and water (2:2:1) and help the phase separation [43]. The reaction mixture was vortexed and centrifuged at 70 × g for 10 min for full phase separation.
The lower layer (i.e. chloroform layer) was collected in a
clean vial. The residue was weighed after evaporating the
solvent at 50°C under the pure nitrogen stream. The lipid
content was measured in duplicate for each sample.
Fatty acid composition
Fatty acid composition of lipid fraction from durian
seed gum was analyzed by using gas chromatography
(Hewlett–Packard GC 6890, Chicago, USA) equipped
with a flame ionization detector (FID) and a fused silica
capillary DB-Wax column (30 mm × 0.32 mm i.d. ×
0.25 lm film thickness) (Agilent Technologies, Chicago,
USA). Fatty acid methyl esters were prepared by using
sodium methoxide and methanol [44]. The injection condition was split mode with the split ratio of 1:10 and injection volume of 2 μl sample. The injector port and
detector temperature were set at 240°C and 270°C, respectively. Helium was used as a carrier gas with the flow
rate of 0.4 ml/min under the constant flow. The oven
temperature was initially set at 80°C, subsequently raised
to 170°C at the flow rate of 10°C/min and kept for 5 min at
170°C. The temperature was subsequently raised to 210°C
at the flow rate of 2°C/min and held for 2 min. Finally, it
was raised up to 240°C at the flow rate of 5°C/min and
kept for 15 min at 240°C. Peak retention times and area
percentages of total fatty acids were determined by injecting
known amount of FAME standards, and subsequently analyzed with the Agilent Technologies ChemStation A.08.03
software. The fatty acid analysis was performed in triplicate
for each sample.
Amino acid analysis
For amino acid analysis, 0.25 g durian seed gum was
weighed and hydrolyzed by using 15 ml 6 N HCl. Then,
it was mixed in a test tube and kept for 24 h at 110°C.
The hydrolysate was dried under the vacuum condition.
Page 12 of 14
The derivatization was carried out by using phenylisothiocyanate. In this experiment, buffer A (0.1 M ammonium acetate, pH 6.5) and buffer B (0.1 M ammonium
acetate, acetonitril, and methanol, 44:46:10 v/v, pH 6.5)
were used as mobile phases. For HPLC analysis, 40 μL of
the sample containing the mobile phase A was injected
into the HPLC system equipped with Photodiode Array
Detector (model MD-2010; JASCO, Tokyo, Japan) and
reversed phase column RP-C18 (LICHROCART 250–
4,6, 250 × 5 mm) (Merck, Darmstadt, Germany). Alpha
amino butyric acid (AABA) was used as an internal
standard (IS). The linear gradient system was used at
the flow-rate of 1 ml/min in an oven at 40°C. The UV
absorption detection at a wavelength of 254 nm was
applied to detect the content of amino acids. The amino
acid analysis was performed in triplicate for each sample.
The result was analyzed by using JASCO Borwin chromatography software (V. 1.5, Jasco Co. Ltd., Japan).
Molecular weight
In the present study, size-exclusion chromatography
coupled to multi angle laser light-scattering (SEC-MALS)
system was applied to measure the molecular weight
according to the method reported by previous researchers
[45]. It should be noted that the multi angle laser light
scattering (MALLS) is for the determination of molecular
weight over broad ranges. The system was equipped
with one guard column (TSK-G), a JASCO PU-980
HPLC pump (Tokyo, Japan) and two separation columns
(TSKgel G-6000 PWxL and TSKgel GMPWxL) (Tosoh
Co., Tokyo, Japan). Exclusion limits of the separation
columns were both 50.0 × 106 g/mol on a dextran base.
Air bubbles of the aqueous solutions containing 0.05 M
NaNO3 were removed by using an on-line degasser
JASCO DG-980-50. Detectors were calibrated by using
filtered toluene and normalized with pullulan (23.8 K)
(Polymer Standards Services GmbH, Mainz, Germany).
A circulating flow rate of 0.5 ml/min was applied in
the system. Static light-scattering measurements using
a DAWN-DSP (Wyatt Technology Co., CA, USA) were
carried out at 25°C, and scattering intensity was determined at angles from 261 to 1321 concurrently with multiple detectors. In this experiment, 0.5 g durian seed gum
was used to prepare the gum solution (0.05%), and the
solution was filtered through ADVANTEC celluloseacetate membrane filters (0.45 mm pore size). Finally,
100 μl of the solution (0.05%) was injected to the system
after filtering through the cellulose-acetate membrane.
The molecular weight was calculated based on the following equations [46]:
D E
i
KC
1 h
¼
1 þ 16π 2 rg2 sin2 ðθÞ=3λ2 þ 2A2 C
Rθ
Mw
ð4Þ
Amid et al. Chemistry Central Journal 2012, 6:117
http://journal.chemistrycentral.com/content/6/1/117
K ¼ 4π 2 no ðdn=dcÞ2 =λ4 NA
X
ðci Mi Þ
Mw ¼ X
ci
X
ci
Mw ¼ X ci
Mi
X
D E
ci Mi 〈r 2 〉i
2
r
¼ X
z
ðci Mi Þ
Page 13 of 14
ð5Þ
ð6Þ
Competing interests
The authors declared that they have no competing interest.
Authors’ contributions
BA carried out all the experiments and data analysis. BA also prepared the
drafted manuscript, and all authors read, edited and approved the final
manuscript.
ð7Þ
Acknowledgment
We appreciate for the financial support of current works from RUGS
(02-01-090666RU) and Science Fund (05-01-04-SF1059) projects supported by
Ministry of Higher Education, Malaysia.
ð8Þ
Author details
1
Department of Food Technology, Faculty of Food Science and Technology,
University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2Faculty of
Agriculture, University “Goce Delcev”, Krste Misirkov bb, 2000 Štip,
Macedonia.
K is an optical constant, Rθ is the excess Rayleigh ratio
which is the measured quantity, θ is the scattering angle,
Mw is the average molecular weight, A2 is the second viral
coefficient, and λ is the wavelength of light. The quantities
ci, Mn Mw are the concentration, number average molecular
weight, and molecular weight, respectively [46]. The ratio of
Mw/Mn represents the polydispersity index. The measurement was triplicate for each sample and the average of three
measurements was reported for further data analysis.
Experimental design and data analysis
The effect of different extraction and purification methods
on the chemical and molecular structure of durian seed
gum was investigated by using the completely randomized
design (CRD). Four different purification methods (namely
Method A (isopropanol and ethanol), Method B (isopropanol and acetone), Method C (saturated barium hydroxide)
and Method D (Fehling solution)) were chosen based on
our previous study and literature [19,21,37,39]. The sugar
composition, moisture, ash, lipid content, fatty acid composition, molecular weight (Mw), and other parameters (Mw/Mn
and Rg) related to the molecular structure of durian seed
gum were determined as response variables. The performance of different extraction and purification techniques was
determined by comparing the chemical and molecular
structure of different crude and purified gums. The data was
subjected to one way analysis of variance (ANOVA) to determine the significant (p < 0.05) differences among the
samples. All data analysis was carried out by using Minitab
version 15 (Minitab Inc., PA, USA). Fisher multiple comparison test was used to evaluate significant differences (p <
0.05) between the different purified seed gums as compared
to the control.
Abbreviations
AABA: Alpha amino butyric acid; ANOVA: One way analysis of variance;
AOAC: Association of Official Analytical Chemists; CRD: Completely
randomized design; D: Durio; FAME: Fatty acid methyl ester; FID: flame
ionisation detector; GF: Glass filter; HPLC: High performance liquid
chromatography; i.d: Inner diameter; IS: Internal standard; LBG: Locust bean
gum; Molar: M; Mw: Molecular weight; Mw/Mn: Molecular weight/number
average molecular weight; RP-C: Reversed phase column; SEC-MALS:
Size-exclusion chromatography coupled to multi angle laser light-scattering;
UV: Ultra violet; v/v: Volume/volume; W/S: Water: seed.
Received: 18 July 2012 Accepted: 1 October 2012
Published: 14 October 2012
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doi:10.1186/1752-153X-6-117
Cite this article as: Amid et al.: Chemical composition and molecular
structure of polysaccharide-protein biopolymer from Durio zibethinus
seed: extraction and purification process. Chemistry Central Journal 2012
6:117.
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